1. Field of the Invention
The present invention relates to methods for producing an L-amino acid utilizing a bacterium, in particular, such a method for producing an L-amino acid utilizing a fatty acid or an alcohol such as glycerol as a raw material, and a bacterium used for the method. L-Amino acids are industrially useful as additives for animal feed, components of health food, amino acid infusions, and so forth.
2. Brief Description of the Related Art
In the industrial production of L-amino acids by fermentation, saccharides, i.e., glucose, fructose, sucrose, blackstrap molasses, starch hydrolysate, and so forth, are used as a carbon source. Moreover, methods for producing an L-amino acid using a fatty acid (WO2009/142286), glycerol (U.S. Patent App. Pub. No. 2009093029), or ethanol (WO2008/010565) as a carbon source have also been disclosed.
In various organisms, in general, an electron withdrawn from Acyl-CoA by Acyl-CoA dehydrogenase at the time of fatty acid utilization is transferred to the oxidized electron transferring flavoprotein (ETFox) via FADH2 to form the reduced electron transferring flavoprotein, ETFred, and an electron is further transferred from ETFred to ubiquinone by ETF ubiquinone oxidoreductase, and then to the respiratory chain (Voet, D. et al., 1995, Biochemistry, Second edition, John Wiley & Sons, Inc., New York). However, for bacteria belonging to the family Enterobacteriaceae, electron transferring flavoprotein (ETF) and ETF ubiquinone oxidoreductase have not been reported to date.
Furthermore, it can be estimated from the descriptions of Coves, J. et al., 1993, J. Biol. Chem., 268(25):18604-18609, Fontecave, M. et al., 1987, J. Biol. Chem., 262(25):12325-12331, and so forth, that at the time of the ethanol utilization in bacteria belonging to the family Enterobacteriaceae, an electron is transferred from NADH, which is considered to be abundantly generated in bacterial cells, or from NADH via NADPH to FAD, and a marked amount of FADH2 accumulates as a result.
For Escherichia coli, fixA (yaaQ) and fixB (yaaR) have been reported as genes coding for ETF homologues that can transfer electrons from FADH2 (Tsai, M. H. et al., 1995, Res. Microbiol., 146:397-404). The fixA and fixB genes constitute the fixABCX operon together with the fixC and fixX genes, and expression thereof is induced under anaerobic conditions in the presence of carnitine (Eichler, K. et al., 1995, J. Basic Microbiol., 35:217-227 and Buchet, A. et al., 1998, J. Bacteriol., 180:2599-2608). There is a report suggesting that the proteins encoded by these genes transfer an electron to crotonobetaine reductase which participates in carnitine metabolism (Walt, A. et al., 2002, J. Bacteriol., 184:4044-4047). In addition, it is predicted that fixA encodes the electron transferring flavoprotein β subunit, fixB encodes the α subunit of the same protein, fixC encodes the electron transferring flavoprotein-quinone oxidoreductase, and fixX encodes a ferredoxin-like protein (Buchet, A. et al., 1998, J. Bacteriol., 180:2599-2608). As described above, although there are reports suggesting involvement of fixA, fixB, and fixC in the carnitine metabolism, it has not been reported to date that they participate in the electron transport from Acyl-CoA dehydrogenase to the respiratory chain.
sodA encodes superoxide dismutase, which catalyzes the conversion of superoxide into hydrogen peroxide (Keele, B. B. et al., 1970, J. Biol. Chem., 245 (22):6176-6181). However, there has not been reported a relationship between the superoxide dismutase activity and production of an amino acid.
As a defense system against intracellular hydrogen peroxide in the bacteria belonging to the family Enterobacteriaceae, the action of the transcription factor OxyR has been reported, which activates the genes for eliminating oxidation stress (Christman, M. F. et al., 1989, Proc. Natl. Acad. Sci. USA., 86(10):3484-3488). It has been reported that, among the genes activated by OxyR, the low molecular RNA coding gene oxyS controls secretion of hydrogen peroxide from bacterial cells (Gonzalez-Flecha, B. et al., 1999, J. Bacteriol., 181(12):3833-3836). However, there has not been reported production of L-amino acid using a microorganism in which oxyS gene expression is enhanced.
It has been reported that thiourea has an effect of suppressing oxidation stress including hydrogen peroxide for Escherichia coli (Kohanski, M. A. et al., 2010, Molecular Cell, 37:311-320). However, any relation between thiourea and L-amino acid production is not known.